Methods for selective targeting

ABSTRACT

A selective targeting method is disclosed which comprises contacting a peptide library with an anti-target to allow the peptides to bind; separating non-binding peptides from the anti-target bound peptides, contacting the non-binding anti-target peptides with a target allowing the unbound peptides to bind with the target to form a target-bound peptide complex; separating the target-bound peptide complex from peptides which do not bind to the target, and identifying the target-bound peptides. In one embodiment the target is skin or hair. In another embodiment the anti-target is hair when the target is skin, and the anti-target is skin when the target is hair.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. Ser. No. 11/588,929,filed on Oct. 27, 2006, now abandoned, which is a divisional of U.S.Ser. No. 10/303,331, filed on Nov. 25, 2002, now U.S. Pat. No.7,129,326, which is a continuation-in-part of U.S. Ser. No. 09/832,723,filed Apr. 11, 2001, now abandoned, which claims the benefit of priorityof U.S. Ser. No. 60/197,259, filed Apr. 14, 2000.

SEQUENCE LISTING

The sequence listing submitted via EFS, in compliance with 37 C.F.R.§1.52(e), is incorporated herein by reference. The sequence listing textfile submitted via EFS contains the file “GC617-3-seqlist” created onMar. 31, 2010, which is 28,672 bytes in size.

BACKGROUND OF THE INVENTION

The present invention is directed to methods for the selection andidentification of compounds capable of binding specifically to a targetin the presence of undesired background targets (anti-targets) usinglibraries of similar compounds. In one particular aspect, the presentinvention is related to the selection of ligands from peptide libraries.Ligand peptides identified according to the method of the invention mayhave a binding affinity and a selectivity to a target similar to thebinding affinity and selectivity of antibodies.

The literature is replete with examples of recent advances in methodsfor screening large library pools of compounds, especially peptides.Methods for screening these compounds to identify molecules that bind toa preselected target have also been advanced. One well-known method isbiopanning which was originally developed by Smith, G. P., (1985),Science 228:1315. Biopanning in its simplest form is an in vitroselection process in which a library of phage-displayed peptides isincubated with a target. The target and phage are allowed to bind andunbound phage are washed away. The specifically bound phage are thenacid eluted. The eluted pool of phage is amplified in vivo and theprocess is repeated. After a number of rounds individual clones areisolated and sequenced.

A number of variations of the biopanning technique first introduced bySmith have been described, and reference is made to Christian et al.,(1992) J. Mol. Biol., 227:711; Cwirla et al., (1990) Proc. Natl. Acad.Sci. USA, 87:6378; Cull et al., (1992) Proc. Natl. Acad. Sci. USA,89:1865; Huls et al., (1996) Nature Biotechnol., 7:276; and Bartoli etal., (1998) Nature Biotechnol., 16:1068.

Huls et al., 1996 supra, describe a method comprising flowcytometry-based subtractive selection of phage antibody on intact tumorcells. The phage-displayed antibodies remain bound to the target duringthe flow-cytometric selection. However, prior to amplification thecell-bound phages are eluted from the target. WO 98/54312 disclosesselection of antibodies under mild conditions with high affinities forantigens using antibody libraries displayed on ribosomes.

In many prior art methods it is generally assumed that elution of targetbound ligands is sufficient to identify the tightest binding ligands ina library. However, a number of research papers report on low affinitybinders using elution techniques (U.S. Pat. No. 5,582,981).Nevertheless, physical separation of the ligands from the target priorto amplification or identification is the standard method for selectingligands that bind to a preselected target.

Balass et al., (1996) Anal. Biochem., 243:264, describe the selection ofhigh-affinity phage-peptides from phage-peptide libraries using abiotinylated target immobilized on a nitrostreptavidin matrix. Theinteracting phage particles were released under conventional acidelution. Further, after acid elution, the target complex was analyzedfor bound phage. These particles were exposed to alkaline solutions orfree biotin to release the target bound phage particles from the solidsupport. The affinity of the isolated phage was found to be higher thanthe phage released by traditional acid elution methods. However, thesynthetically prepared peptides exhibited a lower affinity for thetarget than the peptides prepared from sequences obtained by acid-elutedphage.

Other targeting methods include, for example, SELEX. This is a procedurein which an oligonucleotide from a library of randomized sequences isembedded in a pool of nucleic acids. Many cycles of affinity selectionto a target of the oligonucleotide from the heterologous RNA or DNApopulation occurs. The target and annealed nucleic acids are partitionedand amplified. In order to proceed to the amplification step, selectednucleic acids must be released from the target after partitioning. (U.S.Pat. No. 5,475,096)

While various methods for screening and selecting libraries of compoundsexist, improved methods that do not require multiple rounds of selectionare particularly needed for compounds that a) bind tightly andspecifically to targets that are not well-defined at the chemical,biochemical or genetic level but have macroscopic properties that aredesirable to target, b) bind tightly and specifically to targets thatcannot be easily physically separated from a large background ofundesirable targets (anti-targets), and c) bind to targets under harshconditions, such as acidic pH, high detergent concentration or hightemperature.

The selective targeting method according to the invention overcomes someof the above deficiencies of the prior art methods and in particularoffers an advantage in rapidly identifying compounds, particularlypeptides, that bind with a high affinity and selectively to a target.

SUMMARY OF THE INVENTION

In one aspect, the invention concerns a method for screening a ligandlibrary, particularly a peptide library comprising contacting the ligandlibrary with an anti-target to allow the ligands to bind with theanti-target; separating unbound ligands and contacting said unboundligands with the selected target to allow said unbound ligands to bindwith the target to form a target-bound ligand complex; separating saidtarget-bound ligand complex from ligands which do not bind to saidtarget; and identifying the target-bound ligands on the target-boundligand complex.

In a second aspect, the invention concerns a method for screening aligand library, particularly a peptide library comprising contacting theligand library essentially simultaneously with a selected target and ananti-target to allow the ligands to bind with the target forming atarget-bound ligand complex; separating the target-bound ligand complexfrom the anti-target, anti-target bound ligands and free ligands; andidentifying the ligands of the target-bound ligand complex. Thecontacting step may be accomplished either in vivo or in vitro.

In one embodiment, the anti-target is skin and the target is hair. In asecond embodiment, the anti-target is hair and the target is skin. In athird embodiment, the ligand is a peptide but not an antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a general schematic diagram of the selective targeting methoddisclosed herein. The method comprises the steps of, a) selectionagainst anti-targets which provides a library of ligands depleted ofanti-target bound ligands, b) selection for the target by formation of atarget-bound ligand complex, c) separation of the target-bound ligandcomplex, d) identification of the target-bound ligands, and e)optionally sequencing the target-bound ligands, exposing thetarget-bound ligands to additional rounds of selective targeting, and/ordiversification.

FIG. 2 illustrates enzyme-linked binding assay results for 2 peptides;LESTPKMK (SEQ ID NO. 6) which selectively targets hair and FTQSLPR (SEQID NO. 5) which selectively targets skin (▪ depicts hair and v depictsskin).

DETAILED DESCRIPTION OF THE INVENTION

A. Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention pertains. For the purposes ofthe present invention, the following terms are used to describe theinvention herein.

The term “ligand” refers to a molecule or compound that is recognized bya particular target or anti-target. The term is independent of molecularsize or compositional feature. The ligand may serve as a substrate foran enzyme-catalyzed reaction, as an agonist, as an antagonist, act as asignal messenger, or stimulate or inhibit metabolic pathways. Ligandsmay be nucleic acids, peptides, peptide derivatives, peptidomimetics,polypeptides, small organic molecules, carbohydrates and other moleculesthat are isolated from a candidate mixture that acts on a target in adesirable manner. Preferably the desirable manner is binding the target,but could include for example, catalytically changing the target orreacting with the target that modifies or alters the target.

The term “library” refers to a collection of chemical or biologicalentities that can be created in a single reservoir and simultaneouslyscreened for a desired property. As used herein a library can have aminimum size of at least two members and may contain as many as 10¹⁵members or more. In one aspect, the library has at least 10² members. Inanother aspect, the library has at least 10³ members. In yet anotheraspect, the library has at least 10⁶ members. In a further aspect, thelibrary has at least 10⁹ members. The size of a library refers to thetotal number of entities comprising the library whether the members arethe same or different.

A “peptide library” refers to a set of peptides and to the peptides andany fusion proteins containing those peptides. Stochastic or randomprocesses may be used to construct random peptides. The term “random”does not mean that the library composition is not known.

The term “peptide” refers to an oligomer in which the monomeric unitsare amino acids (typically, but not limited to L-amino acids) linked byan amide bond. Peptides may be two or more amino acids in length.Peptides identified according to the invention are preferably less than50 amino acids in length, more preferably less than 30 amino acids inlength, also preferably less than 25 amino acids in length, andpreferably less than 20 amino acids in length. In one embodiment thepeptides identified according to the method of the invention are between4 and 20 and also between 6 and 15 amino acids in length. However, ingeneral peptides may be up to 100 amino acids in length. Peptides thatare longer than 100 amino acids in length are generally referred to aspolypeptides. Standard abbreviations for amino acids are used herein.(See Singleton et al., (1987) Dictionary of Microbiology and MolecularBiology, Second Ed., page 35, incorporated herein by reference).

The peptides or polypeptides may be provided as a fusion peptide orprotein. Peptides include synthetic peptide analogs wherein the aminoacid sequence is known. The term peptide does not include moleculesstructurally related to peptides, such as peptide derivatives orpeptidomimetics whose structure cannot be determined by standardsequencing methodologies, but rather must be determined by more complexmethodologies such as mass spectrometric methods. Peptidomimetics (alsoknown as peptide mimetics) are peptide analogs but are non-peptidecompounds. Usually one or more peptide linkages are optionally replaced.(Evans et al., (1987) J. Med. Chem. 30:1229). The term “protein” is wellknown and refers to a large polypeptide.

A “skin or hair binding peptide” according to the invention is a peptidethat binds to a target with a binding affinity of at least about 10⁻²M,at least about 10⁻³M, at least about 10⁻⁴ M, at least about 10⁻⁵ M, atleast about 10⁻⁷M, at least about 10⁻⁹M, and preferably between about10⁻² M to 10⁻¹⁵ M, between about 10⁻²M to 10⁻¹⁰M, between about 10⁻³M to10⁻⁹M and between 10⁻⁷ to 10⁻¹⁵M.

The term “nucleic acid” means DNA, RNA, single-stranded ordouble-stranded and chemical modifications thereof. Modifications mayinclude but are not limited to modified bases, backbone modifications,methylations, unusual base pairing modifications, and cappingmodifications. When a nucleic acid library is used in the selectivetargeting method of the invention, the nucleic acid ligand is generallybetween 4 and 250 nucleotides in length, and preferably between 4 and 60nucleotides in length.

The invention further includes ligands, preferably nucleic acid, peptideor polypeptide ligands and more preferably peptide ligands that havesubstantially the same ability to bind to a target as the nucleic acid,peptide or polypeptide identified by the selective targeting methoddescribed herein. Substantially the same ability to bind a target meansthe affinity and selectivity is approximately the same as the affinityand selectivity of the ligands selected by the method herein claimed.

Additionally a ligand having substantially the same ability to bind to atarget will be substantially homologous to the ligand identified by thedisclosed selective targeting method. With respect to a nucleic acidsequence, substantially homologous to an identified ligand means thedegree of primary sequence homology is in excess of 80%, preferably inexcess of 85%, more preferably in excess of 90%, further preferably inexcess of 95%, even more preferably in excess of 97%, and mostpreferably in excess of 99%. It will be appreciated by those skilled inthe art that as a result of the degeneracy of the genetic code, amultitude of peptide encoding nucleotide sequences may be produced. Apeptide or polypeptide is substantially homologous to a referencepeptide or polypeptide if it has at least 50%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90% and at least 95% sequence identity to the reference sequence whenoptimally aligned. Optimal alignment of the sequences may be conductedby various known methods and computerized implementation of knownalgorithms (e.g. TFASTA, BESTFIT, in the Wisconsin Genetics SoftwarePackage, Release 7.0, Genetics Computer Group, Madison, Wis.). Generalcategories of equivalent amino acids include 1) glutamic acid andaspartic acid; 2) lysine, arginine, and histidine; 3) alanine, valine,leucine, and isoleucine; 4) asgaragine and glutamine; 5) threonine andserine; 6) phenylalaine, tyrosine and tryptophan; and 7) glycine andalanine. It is well within the ordinary skill of those in the art todetermine whether a given sequence substantially homologous to thoseidentified herein have substantially the same ability to bind a target.

A small organic molecule as defined herein is a molecule, preferably anonpolymeric molecule, having a molecular weight of approximately 1000Daltons or less and more preferably 500 Daltons or less. A “peptoid” isdefined herein as an enzymatically resistant peptide analog.

The term “target” or “anti-target” refers to molecules or heterogeneousmolecules that have a binding affinity as defined herein, for a givenligand. Both target and anti-targets may be naturally occurring orsynthetic molecules or heterogeneous molecules. In a preferredembodiment, the target is skin or hair. Further when the target is skin,the anti-target is hair, and when the target is hair, the anti-target isskin.

The binding affinity of a ligand for its target or anti-target may bedescribed by the dissociation constant (KO, concentration needed for 50%effective binding (EC₅₀), or concentration needed for 50% inhibition ofbinding of another compound that binds to the target (IC₅₀). K_(D) isdefined by k_(off)/k_(on). The k_(off) value defines the rate at whichthe target-ligand complex breaks apart or separates. This term issometimes referred to in the art as the kinetic stability of thetarget-ligand complex or the ratio of any other measurable quantity thatreflects the ratio of binding affinities, such as an enzyme-linkedimmunosorbent assay (ELISA) signal or radio-active label signal.Selectivity is defined by the ratio of binding affinities or k_(off) fordissociation of the ligand-complex (target K_(D)/anti-target K_(D)). Thek_(on) value describes the rate at which the target and ligand combineto form the target-ligand complex.

The term “contacting” is broadly defined to mean placing a library ofligands and a target or anti-target in immediate proximity orassociation and includes in vitro and in vivo contact. The term includestouching, associating, joining, combining, intravenous injection, oraladministration, intraperitoneally, topical application, intramuscular,inhalation, subcutaneous application and the like. The term “separating”as used herein means to select, segregate, partition, isolate, collect,keep apart and disunite.

“Amplifying” means a process or combination of process steps thatincreases the amount or number of copies of a molecule or class ofmolecules. In one aspect, amplification refers to the production ofadditional copies of nucleic acid sequences that is carried out usingpolymerase chain reaction (PCR) technology well known in the art. Inanother aspect, amplification refers to production of phage virions byinfection of a host.

As used in the specification and claims, the singular “a”, “an” and“the” include the plural references unless the context clearly dictatesotherwise. For example, the term “a ligand” may include a plurality ofligands.

The following references describe the general techniques employedherein: Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Innis etal., PCR Protocols—A Guide to Methods and Applications (1990), AcademicPress, Inc.; Kay et al., (1996) Phage Display of Peptides and Proteins,Academic Press; Ausubel et al., (1987) Current Protocols in MolecularBiology, Greene-Publishing & Wiley Interscience NY (Supplemented through1999); Berger and Kimmel, (1987) Methods in Enzymology, Vol. 152.Academic Press Inc., San Diego, Calif.

The contents of all references, patents and published patentapplications cited throughout this application are hereby incorporatedby reference in their entirety.

B. General Method

Described herein is a selective targeting method for screening a libraryof ligands having a binding affinity and selectivity for a selectedtarget. In its most basic form the selective targeting method may bedefined as follows: Preparing or obtaining a library of ligands,preferably peptides of different sequences and more preferably a randompeptide library. Deselecting ligands that bind with an anti-target bycontacting the ligand library with an anti-target under conditionsfavorable for binding between the ligands of the library and theanti-target; allowing the anti-target to bind with the ligands; andseparating the anti-target non-binders (unbound ligands) from theanti-target ligand bound molecules and any free ligands. Contacting theanti-target non-binders with a selected target under suitable conditionsand allowing them to bind. Ligands with an affinity for the target willbind to form a target-bound ligand complex. The removal of ligands boundto the anti-target and removal of weak target-bound ligands maygenerally be referred to as library depletion. The target-bound ligandcomplex is then separated from the remaining mixture including theunbound ligands, and the target-bound ligands are identified. Thetarget-bound ligand complex or the target-bound ligands may thenoptionally be subjected to amplification, sequencing or further roundsof selection (FIG. 1). The invention further comprises the ligandsidentified according to the selective targeting method of the invention.

In the practice of the invention, a library of compounds to be testedwill generally be provided. A library of ligands may include, but is notlimited to, random peptide libraries, synthetic peptide orpeptidomimetic combinatorial libraries, peptide loop libraries,combinatorial chemical libraries, and oligonucleotide libraries. Theselibraries are well known to those in the art as well as methods formaking said libraries. Reference is made to Barbas, C. F. (1993) CurrentOpinion in Biotech., 4:526; Cwirla et al., (1990) supra; Scott andSmith, (1990) Science, 249:386; Cull et al., (1992) supra; Pinilla etal., (1994) Biochem. J. 301:847; Sambrook et al., (1989) supra; Ausubelet al., (1987) supra; and Gubler and Hoffman, (1983) Gene 25:263; eachof which is incorporated herein by reference.

One preferred type of library includes random peptide libraries (alsosometimes referred to in the art as epitope libraries). These librariesmay include cell-surface display libraries, for example yeast display(Boder and Wittrup (1997) Nat. Biotechnol., 15:553); peptide librariesinserted into proteins (Lenstra et al., (1992) J. Immunol. Methods,152:149 and U.S. Pat. No. 5,837,500); direct screening of peptides onpolysomes (Tuerk et al., (1990) Science 249:505) and phage displaylibraries (Delvin et al., (1990) Science 249:404; WO91/18980; Dower etal. WO91/19818; and Parmley et al., (1988) Gene 73:305). Phage displaylibraries are particularly preferred. A phage display library is alibrary in which numerous peptides are displayed on the surface of abacteriophage, such as a filamentous phage. The peptide or protein isexpressed as a fusion with a coat protein of the bacteriophage resultingin display of the fusion protein on the surface of the virion while theDNA encoding the fusion resides within the virion. Suitable non-limitingexamples of vectors for construction of phage libraries include fAFF1;the fUSE series, such as fUSE5; lamba phage vectors; and T7select(non-filamentous) phage vectors. (Smith and Scott (1993) MethodsEnzymol. 217:228; and Cwirla et al., (1990) Proc. Natl. Acad. Sci. USA87:6378). Phage-peptide library kits are available and reference is madeto Chiron Corp. (Emeryville, Calif.), New England BioLabs Inc., CatalogNo. 8100 (Beverly, Mass.), and Novagen Catalog No. 70550-3 (MadisonWis.). While various antibody libraries are known, including antibodydisplay libraries on phage (de Bruin et al., (1999) Nat. Biotechnol.,17:397), in one preferred aspect of the present invention, the libraryof ligands used in the selective targeting method according to theinvention will not include antibodies.

Another type of peptide library encoded by nucleic acids includes alibrary wherein the peptide is expressed as a fusion with anotherprotein, for example, either a cell-surface protein or an internalprotein of a host. The nucleotides encoding the peptide are insertedinto a gene encoding the internal protein. Various examples of this typeof library include the fusion of peptides to a lac repressor, GAL4,thioredoxin, and various antibodies (U.S. Pat. Nos. 5,283,173;5,270,181; and 5,292,646). Cull et al. (1992) Proc. Natl. Acad. Sci. USA89:1865 teach the construction of a fusion gene encoding a fusionprotein of peptide library members and Lacl. Nucleic acids encoding alibrary of peptides are inserted into a gene encoding Lacl. The fusionprotein and the fusion plasmid encoding the fusion protein arephysically linked by binding of the peptides to the lac operatorsequence in a plasmid. Host cells may be transformed with the libraryplasmids. The cells expressing the fusion protein are lysed releasingthe fusion protein and associated DNA (see for example U.S. Pat. No.5,733,731). The library can then be screened or selected. DNA shuffledlibraries are also known which are constructed by homologous exchange ofDNA fragments during DNA recombination methods or by synthetic methods(see for example U.S. Pat. No. 5,605,793 and Stemmer (1994), Proc. Natl.Aca. Sci. USA 91:10747).

So called anchor libraries have been described in PCT US96/09383 and WO97/22617. This is a peptide library wherein peptides have non-continuousregions of random amino acids separated by specifically designated aminoacids. These libraries are made by genetic or chemical means.

A combinatorial chemical library and particularly a peptide library mayalso be synthesized directly by methods known in the art including, butnot limited to synthesis by arrays (Foder et al., (1991) Science251:767); synthesis on solid supports (WO97/35198); and other chemicalmethods such as those disclosed in Lam et al., (1993) Bioorg. Med. Chem.Lett., 3:419, Tjoeng et al., (1990) Int. J. Pept. Protein Res. 35:141,and WO96/33010.

Methods for creating combinatorial chemical libraries are also known inthe art. Combinatorial libraries include large numbers of chemicalvariants for peptides, oligonucleotides, peptoids, carbohydrates, smallorganic molecules and even solid-state materials (Schultz et al., (1995)Science, 268:1738). A core structure will be varied by addingsubstituents or by linking different molecular building blocks.Libraries may include molecules free in solution, linked to solidparticles or beads, or arrayed on surfaces of modified organisms.Varying substituents around the core molecule may modify virtually anyclass of compounds. Various non-limiting examples of classes ofcompounds for combinatorial libraries include benzodiazepines;mercaptoacyl prolines; carbamates; chalcone libraries; ketoamideconjugates; polyketones; paclitaxel libraries; anilides;aryloxyphenoxypropionates; oxazolidinones; carbohydrates; and numerousother classes. While methods for making combinatorial libraries are welldocumented in the literature, these methods may be very time consuming.Various companies now make instrumentation to generate combinatoriallibraries from both solution and solid phase synthesis (CombiChem. Inc.(San Diego, Calif.); Advanced ChemTech (Louisville); Zymark Corp. (MA);and Hewlett Packard (CA)). Once a library has been generated it canoptionally be purified for example by high performance liquidchromatography (HPLC). Once a small organic molecule is screened andidentified according to the selective targeting method of the invention,it may be produced on a larger scale by means of organic synthesis knownin the art.

As taught herein not only are standard methods for generating librariesof ligands well known, but also ligand libraries may be obtainedcommercially, for example from Sigma (St. Louis Mo.) or from variouspublic sources such as American Type Culture Collection (ATCC) and theNational Institute of Health (NIH).

Suitable targets and anti-targets used in the selective targeting methodaccording to the invention include, but are not limited to, proteins,peptides, nucleic acids, carbohydrates, lipids, polysaccharides,glycoproteins, hormones, receptors, antigens, antibodies, viruses,pathogens, toxic substances, metabolites, inhibitors, drugs, dyes,nutrients, growth factors, cells or tissues.

Sources of cells or tissues include human, animal, bacterial, fungal,viral and plant. Tissues are complex targets and refer to a single celltype, a collection of cell types or an aggregate of cells generally of aparticular kind. Tissue may be intact or modified. General classes oftissue in humans include but are not limited to epithelial, connectivetissue, nerve tissue, and muscle tissue.

It is preferred that the target and anti-target are characterized insome detail at the structural, chemical or genetic level to allow somecontrol over the purity, stability and concentration of the target.However, targets and anti-targets may be used that are not wellcharacterized. Non-limiting examples of potentially notwell-characterized targets include collar soil, tumor cells, human skinand hair.

In another aspect, when the target is damaged cells, tissue, or organs,the anti-target is healthy normal (non-damaged) cells, tissue, organs orcombinations thereof. Specific non-limiting anti-target examples includehealthy normal whole blood, skin, hair, teeth, and nails.

In some applications, the target and anti-target can be reverseddepending upon the specific application of interest. For example theremay be multiple applications where it is desirable to target human skinand not hair. Therefore the anti-target would be hair. In a similarapplication it may be desirable to target human hair and not thecorresponding anti-target, skin.

The following general examples of target/anti-target used in the sameapplication are provided for illustrative purpose only and are not meantto limit the selective targeting method disclosed herein: tumorcell/normal cell; receptor cell/cell not expressing the receptor;neoplastic cell/normal cell; soil stain/cotton fabric; foodstain/ceramic; specific protease/other protease; serine protease/wholeblood; hematopoietic stem cell/whole blood; specific enzymevariant/other forms of the enzyme; virus in a cell/cell; TNF-alpha/bloodcomponents; specific insect enzyme/homologous enzymes in animals;hematopoietic stem cell/other hematopoietic cells; hair/skin;nucleus/mitochondria; cytoplasm/nucleus; alpha/beta hydrolases/otherhydrolases; and a specific enzyme involved in photosynthesis/leaftissue.

Both the target and anti-target concentrations to be used in theselective targeting method will vary depending on the type of ligandlibrary, anti-target and target used. As discussed herein, the disclosedmethod has wide applicability to many different targets andanti-targets, therefore the concentration useful in the method may varyfrom about 1.0 M to 10⁻¹⁵ M, preferably the concentration is in the 10⁻⁹M range. In general an excess amount of anti-target relative to theamount of target is required. While not meant to limit the invention,this excess amount may be in the range of at least 10 fold greater tomore than 1000 fold greater. An initial target concentration may bepreferably provided in the range of 10⁻³ M to 10⁻¹⁵ M. In oneembodiment, when the target is an enzyme, the target concentration maybe provided in the range of about 10⁻³ M to 10⁻¹² M. In anotherembodiment, when the target is a cytokine, the target may be provided inthe concentration range of about 10⁻³ M to 10⁻¹² M. In yet anotherembodiment, when the target is a hematopoietic cell, the targetconcentration may be provided in the range of about 10 to 10⁹ cells.

In certain preferred embodiments, the anti-target or target may be amaterial or surface, such as a fabric, ceramic, micro-fluidic chip, skinor hair. In this instance the area of the target or anti-target will beimportant. While not intended to limit the invention in any manner, ingeneral the size of the anti-target or target material will be about 1.0mm to 1.5 cm; more preferably about 25.0 mm to 0.5 cm; however, thediameter or area may be more or less than these values. While not meantto limit the invention, when human hair is a target or an anti-targetthe diameter of the hair strand may generally be between about 10 to 220μm, between about 15 to 190 μm and between about 70 to 115 μm. When skinis a target or anti-target, typical skin swatches used in the disclosedmethod are between about 0.2 to 8.0 cm² and also between about 0.4 to4.0 cm².

In one aspect, the invention is directed to the screening andidentification of ligands that bind to a selected target to form anon-covalent target-ligand complex with a binding affinity in the rangeof antibody affinities for antigens. The ligand binding affinityaccording to the present invention for K_(D), EC₅₀ or IC₅₀ is in therange of between about 10⁻²M to 10⁻¹⁵M, about 10⁻²M to 10⁻¹⁰M and about10⁻⁷M to 10⁻¹⁵M, although higher or low binding affinities may beachieved. In one aspect, the binding affinity is in the range of atleast about 10⁻²M, at least about 10⁻³M, at least about 10⁻⁴M, at leastabout 10⁻⁵M, at least about 10⁻⁷M, at least about 10⁻⁹M and also atleast about 10⁻¹² M. In another embodiment, the affinity is less thanabout 10⁻⁷M. In another aspect, k_(off) values for the ligand-targetcomplex will be less than about 10⁻² sec⁻¹, less than about 10⁻³ sec⁻¹,less than about 10⁻⁴ sec⁻¹, and also less than about 10⁻⁵ sec⁻¹. Theligands identified according to the selective targeting method of theinvention will not bind with any significance to the anti-target. Whilenot meant to limit the invention, a preferred ligand identifiedaccording to the selective targeting method described herein may have aK_(D) for the anti-target greater than about 10⁻⁴ M, and preferablygreater than about 10⁻¹ M.

The selective targeting method according to the invention may becharacterized not only by the binding affinity of a ligand to thetarget, but also may be characterized by the selectivity of theligand-target complex. The selectivity of ligand binding for a targetcompared to ligand binding to an anti-target can be defined by a ratioof K_(D), EC₅₀ or IC₅₀ in the range of about 2:1 to 500:1. In oneaspect, selectivity is at least about 2:1, at least about 3:1, at leastabout 5:1, at least about 10:1, at least about 20:1, at least about30:1, at least about 50:1, and at least about 100:1.

In another aspect, the selective targeting method may be used to selectligands with a low affinity for the target but with a high selectivityfor the target. In this aspect, the selectivity of ligand bindingaffinity for the target compared to said ligand binding to ananti-target would be at least about 2:1, at least about 3:1, at leastabout 5:1, at least about 10:1, also at least about 20:1, at least about50:1, and even at least about 100: or greater. However, the targetbinding affinity would be in the range of about 10⁻²M to 10⁻⁷M, in therange of about 10⁻²M to 10⁻⁵ and also in the range of about 10⁻²M to10⁻³.

Methods for measuring binding affinities and selectivity are well knownin the art, and these methods include but are not limited to measurementby radio-labeled release and competition assay; by isothermal titrationcalorimetry; biosensor binding assays (Morton & Myszka, (1998) MethodsEnzymol. 295:268-294); by fluorescence and chemi-luminescencespectroscopy; and by mass spectrophotometry (Gao et al., (1996), J. Med,Chem., 39:1949).

In one aspect, the anti-target is combined with the library of ligandsand allowed to incubate prior to exposing the library of ligands to thetarget. In another aspect, the anti-target and target are combined withthe library of ligands essentially simultaneously. Essentiallysimultaneously means at the same time or very close in time wherein theligand library is exposed to both the anti-target and the target priorto any separation step.

The selective targeting method as described herein may be performed invitro or in vivo. When performed in vitro, the library of ligands andthe anti-target (and optionally the target), are combined in or on avessel. The vessel may be any suitable material or receptacle such as aplate, culture tube, micro titer plate, micro-fluidic chip, petri dishand the like.

Preferably, the anti-target and the target are available in anenvironment where non-specific binding events are minimized. This may beaccomplished by various means including, but not limited to, 1) bycoating a vessel containing the ligand library and thetarget/anti-target with BSA, skim-milk or other adsorbing protein toblock non-specific binding, 2) by labeling the target molecule with acapture agent such as a biotinylated compound, for example biotin,avidin, or mutated form thereof which can be subsequently trapped bystreptavidin or a streptavidin derivative, such as nitrostreptavidin, 3)displaying the target/anti-target on magnetic beads that can bephysically separated from the library, or 4) by using library displayvectors with low background adsorption properties. These methods areknown in the art and reference is made to Parmley et al. (1988) supra;and Bayer et al., (1990) Methods Enzymol. 184:138.

A composition including a library of ligands and an anti-target may becombined together with additional compounds such as buffers andoptionally detergents and organic solvents under suitable conditions toallow binding of the ligands with the anti-target. One skilled in theart is well aware of useful buffers. Non-limiting examples include;tris(hydroxymethyl)aminomethane (Tris) buffers;N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) buffers;morphololino-ethanesulfonic acid (MES) buffers; buffered salinesolutions, such as N,N-bis[2-hydroxyethyl]2-aminoethanesulfonic acid(BES), Tris, and phosphate-buffered saline (PBS), preferably bufferedsaline solutions (Sambrook et al., (1989) supra). Commercial buffers areavailable for example SuperBlock™ (Pierce, Rockford, Ill.). Otheringredients such as detergents, for example Tween and Triton can be usedin the solutions.

Depending on the target, the composition including the ligand libraryand anti-target is incubated for a period of about 1 minute to about 96hours to allow the ligands to bind with the anti-target. However, longertime periods may be used depending on the stability of the target,anti-target and ligand library. The component containing the unboundanti-target ligands is separated from the anti-target bound ligandsafter incubation. While not essential, the separated component includingthe unbound anti-target ligands may optionally be transferred to a newvessel including the anti-target, incubated and then the componentcontaining the unbound anti-target ligands can again be separated fromthe bound anti-target ligands. This transfer process may be repeatednumerous times, for example it may be repeated between 2 to 10 times ormore. The repeated transfer step further reduces the number of ligandsthat bind to the anti-target. However, the contacting of the library ofligands with the anti-target and the separating of the anti-target boundligands from the unbound ligands may be accomplished in one round. Thecontacting including incubation, and the separation steps, whethercompleted in one round or in multiple rounds may generally be referredto as deselection.

In general, the temperature conditions during deselection may be between2 and 30° C. The temperature is limited by the stability of thecomponents and is well within the skill of one of ordinary skill in theart to determine.

The unbound anti-target ligands may be separated from the anti-targetbound ligands by methods well known in the art. Some of these methodsinclude liquid transfer, washing, centrifugation, filtration,chromatography, micro-dissection and fluorescence activated cell sorting(FACS).

The ligand library, depleted of anti-target binding ligands andcontaining unbound ligands is transferred to a vessel including thetarget under suitable conditions which will allow one or more members ofthe ligand library to bind with the target thereby forming atarget-bound ligand complex. In one aspect the ligands may be contactedwith the same target. In another aspect the ligands may be contactedwith an array of targets at the same time. One non-limiting example ofan array of targets includes the contacting of a ligand with multiplestains on a surface. The ligands are incubated under conditions thatallow binding to the target and generally for a period of time rangingfrom about 1 minute to about 96 hours. The incubation time depends onthe stability of the target. When the target is a stain, the incubationperiod will generally range from about 5 minutes to about 90 minutes.The vessel may further include buffers as described herein above. Thetemperature range is generally between about 2 and 30° C., andpreferably about 18 to 25° C.

One skilled in the art is well aware of references describing cell,organ, and tissue culture, and reference is made to Atlas and Parks(eds) (1993), The Handbook of Microbiological Media, CRC Press, BocaRaton Fla.; Gamborg and Phillips (eds) (1995) Plant Cell Tissue andOrgan Culture, Fundamental Methods, Springer Lab Manual Springer-Verlag.

The target-bound ligand complex may be subject to one or more washsteps. The washing compounds may include buffers (such as TBS and PBS),detergents, acids (glycine), organic solvents, bases, enzymes,sonication, or combinations thereof, wherein unbound ligands are washed.When the target-bound ligand complex is subject to an acid elution, thepH of the acid elution may be in the range of about 1.5 to 4.5,preferably in the range of about 2.0 to 3.5. The acid elution may takeplace for between 2 to 20 minutes and generally no longer than about 10minutes. The wash step may be repeated numerous times and in general canbe repeated between 2-6 depending on the specific target and ligandlibrary. Particularly when the washing step is with an acid, washingwill generally be followed by neutralization with various well-knowncompounds and buffers, such as TRIS-HCL. The washing step results in atarget-bound ligand complex comprising tight binding ligands having aK_(D), k_(off) and selectivity values as herein defined.

When the ligand library is contacted with the anti-target and targetessentially simultaneously as opposed to sequentially, the ligandlibrary, anti-target and target composition may further include allmaterials described above for the sequential exposure of the anti-targetand target.

Further when the ligand library is contacted with the anti-target andtarget essentially simultaneously, the method may also be performed invivo. In this aspect, the library of ligands may be administered bymeans well known in the art, but preferably by injection into a host. Ifthe library is a phage-peptide library, the number of transducing unitsmay be in the range of 10⁴-10¹⁰. The host may be any animal, such as ahuman, mouse, chicken, or pig, preferably mouse. The target for examplemay be whole organs or damaged or tumor tissue, more specifically tumorblood vessels. If the target is a tissue or cells found in the blood,the library of ligands may be circulated in the blood for a period ofabout 1 minute to 10 minutes and allowed to bind with the target. Thetarget-bound ligand complex may be recovered after perfusion and thetissue dissected (Koivunen et al., (1999) Nature Biotech. 17:768 andArap et al., (1998) Science 279:377).

Separation of the target-bound ligands from the anti-target unboundligands or free ligands in the mixture may also be accomplished bywell-known means in the art and these methods include affinitychromatography; centrifugation; high-performance liquid chromatography(HPLC); filtration, such as gel filtration; enzyme-linked immunosorbentassays (ELISA); and fluorescence-activated cell sorting (FACS). Thechoice of the separating method will depend on various factors such asthe target, anti-target and ligand molecules. The choice of theseparation method is well within the skill of one in the art and avariety of instruments used for these separation methods arecommercially available. (See Kenny and Fowell (eds) (1992) PracticalProtein Chromatography Methods in Molecular Biology, vol. 11, HumanaPress, Totowa N.J.).

The target-bound ligand on the target-bound ligand complex may beidentified by various techniques including polymerase chain reaction(PCR), mass spectrophotometry (MS), surface plasmon resonance,immunoprecipitation and nuclear magnetic resonance (NMR) spectroscopy(U.S. Pat. No. 4,683,202; Szabo et al., (1995) Curr. Opin. Struct. Bio.5:699; Harlow et al., (1999) Using Antibodies, A Laboratory Manual, ColdSpring Harbor Press; and Hajduk et al., (1999) J. Med. Chem., 42:2315).Asymmetric PCR may also be used for identification of the target-boundligand wherein a single primer species or primers in differentialconcentration may be used. As well known to those in the art, when thelibrary members are genetically linked to the peptide or protein, DNA ormRNA can be amplified by PCR and the corresponding sequence subclonedinto a vector for sequencing and identification.

During the process of the identifying step, the target-bound ligand mayseparate from the target-bound ligand complex, but the identifying stepdoes not require separation, and preferably the target-bound ligand isnot separated from the target-bound ligand complex prior toidentification of the ligand. For example, in mass spectrophotometry(MS), once the target-bound ligand complex is injected into the massspectrophotometer the target-bound ligand may be separated from thetarget complex. Additionally, PCR may be directly carried out on thetarget-bound ligand complex.

The selective targeting method according to the invention preferablyincludes PCR to identify target-bound peptides. According to theinvention use of PCR results in the recovery of peptides not recoveredby conventional biopanning methods which utilize acid-elution. Ingeneral, a ligand encoding a DNA is amplified by PCR with appropriateprimers.

The presence of specific PCR products indicates that the target-boundligand encoding DNA is present. The amount of the target-bound ligand isdetermined by quantitative PCR. The degree of wash stringency can bemonitored to a desired level and to very low detection levels forexample to attomole levels. Nonspecific ligand binders may be competedout for example by adding wild type phage and designing primers thatonly amplify the ligand library. To prevent deterioration ofsignal-to-noise ratio, the sequences flanking the ligand encoding DNAmay be changed frequently during rounds of selection. Sensitivity forthe analysis of target-bound ligands may be controlled by changingtarget concentration, the number of PCR amplification cycles, thespecificity of the PCR primers, and the detection method for PCRproducts.

Various inhibitory reactions of PCR may be alleviated by the addition ofexcipients including bovine serum albumin, cationic amines, and organicsolvents and reference is made to Roux, (1995) “Optimization andTroubleshooting in PCR” in PCR Primer: A Laboratory Manual, Cold SpringHarbor Press. DMSO and glycerol may be used to improve amplificationefficiency and specificity of PCR. The DNA of the target-bound ligandmay also be extracted and purified using standard techniques.

To facilitate sequencing of desired clones or separation from undesirednon-specific phage, the polynucleotide products generated by PCR may belabeled for example with biotinyl or fluorescent label moieties byincorporation during polymerase mediated catalysis. When the desired PCRproduct is to be cloned into a vector for additional rounds of selectivetargeting according to the method of the invention, it may be desirableto introduce diversity by mutagenic PCR methods, (See Stemmer, in Kay etal., supra). These include cassette mutagenesis, error prone PCR, DNAshuffling, ITCHY-SCRATCHY and the like as is well known by those in theart. Also reference is made to Tillett and Neilan, (1999) “Enzyme-freeCloning: A Rapid Method to Clone PCR Products Independent of VectorRestriction Enzyme Sites”: Nucl. Acids. Res., 27:26e.

As mentioned above and as well known in the art, the PCR fragments maybe cloned into various vectors for sequencing, they may be used in theformation of peptide protein fusions, or cloned into additional displayvectors.

The target bound library members may also be identified preferably bymass spectrometric methods. This is a rapid and accurate identificationof the structure of a compound based on the mass of the compound and onfragments of the compound generated in the mass spectrometry. The use ofmass spectrometry to identify the structure of compounds has beenreported in Cao et al., (1997) Techniques in Protein Chemistry VIII,Academic Press pages 177-184; and Youngquist et al., (1995) J. Am. Chem.Soc. 117:3900. Also reference is made to Cheng et al., (1995) J. Am.Chem. Soc., 117:8859 and Walk et al., (1999) Angew. Che. Int. Ed.,38:1763. One mass spectrometric technique is tandem mass spectrometry(MS/MS) wherein mass spectrometry is performed in tandem with liquidchromatography. To purify and separate the ligand of interest, this typeof MS is preferably used to screen target-bound ligands other thanphage-type peptides because of the need to separate and purifytarget-bound ligands from a biological system prior to injection of theligands into a mass spectrometer. Various recently developed MStechniques are available for identification of the target-bound ligands.(See Wu et al., (1997) in Chemistry and Biology, vol. 14(9):653,Marshall et al., (1998), Mass Spectrometry Reviews 17:1, and Nelson etal., (1999) J. Mol. Recognition, 12:77).

Following the screening of one or more ligand members, particularlypeptide ligands, the amino acid sequence of the peptides may bedetermined according to standard techniques known by those in the artsuch as direct amino acid sequencing of the selected peptide by usingpeptide sequencers, MS/MS, or manually or by determining the nucleotidesequence that encodes the peptide.

In a particular embodiment, the method concerns selecting peptides froma peptide library having a binding affinity for a target of betweenabout 10⁻²M to about 10⁻¹⁵M and between about 10⁻²M to about 10⁻¹⁰Mwhich comprises, contacting a peptide library with an anti-target toallow the peptides in the library to bind with the anti-target;separating unbound peptides from the anti-target bound peptides;contacting the separated unbound peptides with a target under conditionsallowing binding of the unbound peptides with the target to form atarget-bound peptide complex; separating the target-bound peptidecomplex from the peptides that do not bind to the target; andidentifying the bound peptides on the target-bound peptide complexwherein the bound peptides are less than about 50 amino acids in length,are not antibodies, and have a selectivity in the range of about 2:1 toabout 50:1. Preferably the target is skin or hair, and the anti-targetis skin when the target is hair or the anti-target is hair when thetarget is skin. In one embodiment the bound peptides identified on thetarget-bound peptide complex are less than 25 amino acids in length withselectivity in the range of at least about 2:1 and also at least about5:1.

Once the target-bound ligands are identified, the ligands may be exposedto repeated rounds of the selective targeting method and reference ismade to FIG. 1. The target-bound ligands may be subject todiversification. Diversification including chemical diversity mayinclude a number of mutagenesis techniques. See Saiki et al., (1988)Science 239:487; Zoller et al., (1982) Nucl. Acids. Res. 10:6487; andSmith (1985) Ann Rev. Genetics. 19:423. The target-bound ligands may besequenced to determine the identity of the bound ligands and thenoligonucleotides may be made based on the sequences but which includesmall variations. PCR may be used to make small changes in thenucleotide coding sequences for the ligands. This PCR mutagenesis canresult in a mutation at any position in the coding sequence.Diversification may also take place by mutagenesis of a small subset ofidentified ligands. In general diversified ligands will have at least80%, 85%, 90%, 95%, 97% or 99% sequence identity at the nucleotide levelto the target-bound ligand. When the ligand is a peptide the diversifiedpeptide will have at least 80%, 85%, 90%, 95%, 97% or 99% amino acidsequence identity to the identified target-bound peptide. Thediversified ligands may be exposed to one or more rounds of theselective targeting method of the present invention. The diversifiedligands may be screened with other identified target-bound ligands fromwhich they were derived and assayed in appropriate applications forwhich the ligands were originally screened.

The selective targeting method of the current invention for screening alibrary of ligands that bind to a target has wide utility for manyapplications. In one particular application, the selective targetingmethod described herein may be used to identify ligands that bind to atarget under harsh conditions. A harsh condition may include but is notlimited to acidic pH, high temperature, and exposure to detergents, suchas those found in household laundry detergents. In this respect, oneexemplary application according to the invention is screening andidentification of a ligand, particularly a peptide, which is useful incleaning applications. Cleaning applications include but are not limitedto detergent compositions, stain removal compositions, and textiletreatment compositions. Particular stain targets include human body soilstain, a porphyrin derived stain, a tannin derived stain, a carotenoidpigment derived stain, an anthocyanin pigment derived stain, asoil-based stain, or an oil-based stain. Components of various cleaningcompositions and particularly detergent compositions, are well known inthe art and are not repeated herein. See for example the followingreferences U.S. Pat. Nos. 3,929,678; 4,760,025; 4,800,197; 5,011,681;and McCutheon's Detergents and Emulsifiers, North American Edition(1986) Allured Publishing Co.

In a further application skin or hair binding peptides of the inventionmay be used in compositions for personal care applications. Thesecompositions may take the form of lotions, creams, gels, sprays,shampoos and conditioners and the like.

Non-limiting examples of personal care applications which include abinding peptide of the invention are the following: a) using a skinbinding peptide with an emollient which may result in the enhancement ofthe moisturizing properties of the emollient; b) combining a skinbinding peptide with a bleaching or tanning agent which may result inthe enhancement of skin bleaching or tanning properties; c) combining askin binding peptides with a sunscreen for topical application; and d)combining a hair binding peptide with a dye or oxidizing agent whereinthe hair coloring properties of the hair coloring formulation may beenhanced.

One skilled in the art is aware of various references including lists ofcosmetic raw materials which may be used in personal care compositions.Two such references are CTFA International Buyers' Guide, 2002,Cosmetic, Toiletry and Fragrance Association, Washington D.C. and CTFAInternational Cosmetic Ingredient Dictionary and Handbook, 7th Ed.(1997) Vol. 2, Eds. Wenninger and McEwen, Cosmetic, Toiletry andFragrance Association, Washington D.C. Also reference is made to WO00/24372; WO 96/16630 and Sagarin, Cosmetics, Science and Technology,2nd Ed. Vol. 1 (1972).

The selective targeting method and the ligands identified according tothe method may be used in broad applications. In addition to theapplications discussed herein above, other non-limiting applications,particularly for peptide ligands include: 1) for mapping antibodyepitopes; 2) in providing new ligands for important binding molecules,such as enzymes and hormone receptors; 3) in providing potentialagricultural compounds with pesticidial properties; 4) for developingnew drug leads and exploiting current leads; 5) identifying industrialcatalysts; 6) in identifying highly sensitive in vivo and in vitrodiagnostic agents; 7) for increasing the efficiency of enzyme catalystsby binding metals and other cofactors; 8) for controlling proteaseaction in vivo; 9) to change inhibitory properties of targeted proteins;10) use in developing a targeted enzyme; 11) use in selective deliveryof gene therapy vectors to specific tissues or cell types; and 12) usein drug delivery or targeted actives.

Accordingly, the following examples are offered by way of illustration,and are not meant to limit the invention in any manner. Those skilled inthe art will recognize or be able to ascertain using no more thanroutine experimentation, many equivalents to the specific embodiments ofthe invention described herein.

EXAMPLES

The procedures for restriction digest, ligation, preparation ofcompetent cells using calcium chloride, preparation of 20 mg/mlisopropyl (IPTG), preparation of 20 mg/ml5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal), and preparation ofphosphate-buffered saline (PBS) were according to well-known methods inthe art and can be found in Sambrook et al. (1989) supra.Phage-displayed libraries (cyclic 7-mer, linear 7-mer and linear 12-mer)were supplied by New England Biolabs ((NEB; Beverly, Mass.). Restrictionendonucleases EagI and Acc65I, 10× NEBuffer 3, T4 DNA ligase, alkalinecalf intestinal phosphatase, E. coli ER2537 host strain, and M13KE gIIIcloning vector were supplied by NEB and used according to themanufacturer's instructions unless stated otherwise. Taq polymerase,10×PCR Buffer, and dNTP mix were supplied by Roche MolecularBiochemicals (Indianapolis, Ind.). The HotStart Taq Master Mix kit forPCR came from Qiagen (Valencia, Calif.). PCR was carried out using aHYBAID Omn-E Thermocycler from E&K Scientific Products (Campbell,Calif.) or PTC 2000 DNA Engine™ from M.J. Research Inc. (Roche MolecularSystems, Inc. Alameda, Calif.). Nondenaturing polyacrylamide gels (8%)and D-15 DNA markers were obtained from Novex (San Diego, Calif.) and 2%E-gels and TOPO cloning kits were obtained from Invitrogen (Carlsbad,Calif.). Both the QIAquick Gel Extraction Kit and QIAquick PCRPurification Kit were obtained from QIAGEN (Valencia, Calif.). AmpliWax™PCR Gems were obtained from Perkin Elmer (Boston, Mass.).

Example 1 Screening for Peptides Selected to Target Human Skin and notHair

Two 3 inch strands of dark human hair (International Hair Importers &Products, White Plains, N.Y.) were placed in BSA blocked 50 ml conicaltubes containing 10 ml of a 2% Neutrogena® Bath Gel (Neutrogena Corp.)solution in DI water. 10 μL of cyclic 7-mer or linear 12-mer peptidelibraries (10¹⁰ pfu/μl), or wild type phage (10⁹ pfu/μl) were added andthe samples mixed at room temperature for 15 min with rotatory shaking(30 rpm). The unbound supernatant was transferred to a new tubecontaining an additional two 3 inch strands of dark hair, and incubatedat room temperature for 15 min with rotary shaking. After this secondhair incubation, 500 μl of the solution was transferred to the surfaceof human skin tissues (EpiDerm™, MatTek Corp. Ashland, Mass.) in a 6well culture plate containing 0.9 mL tissue culture media (MatTek Corp)for 30 minutes at room temperature with gentle agitation. The skintissues were removed and washed 2× in 50 mls of 2% bath gel for 5 mineach and 3× in 50 mls of PBS for 5 min each in blocked 50 mL conicaltubes. After the final PBS wash, the skin tissues were frozen at −20° C.followed by PCR of the target bound ligand phage. A skin binding peptideidentified according to this example containing a consensus sequenceTQSL is FTQSLPP (SEQ ID NO. 5).

Example 2 Screening for Peptides Selected to Target Human Hair and NotSkin

Pre-equilibrated skin tissues were placed into a 6 well culture platecontaining fresh 0.9 mL tissue culture media and 300 μl of a 2%Neutrogena® Bath Gel containing, 10 μL of cyclic 7-mer or linear 12-merpeptide libraries (10¹⁰ pfu/μl), or wild type phage (10⁹ pfu/μl) wereadded to the skin surface. The samples were incubated at roomtemperature for 15 min with gentle agitation. The unbound supernatantwas transferred to a new well containing skin tissue and the procedurewas repeated. The incubation solution was transferred to nine 3 inchdark hair (International Hair Importers & Products, White Plains, N.Y.)strands in 50 ml tubes containing 10 ml of 2% body gel for 30 minutes atroom temperature with rotatory shaking (30 rpm). The hair samples werethen washed with 1×50 mls, 2×50 mls, or 4×50 mls of 2% bath gel; Washcycles in PBS followed (1×25 mls for 5 min, 1×25 mls for 2 min, 2×50 mlsfor 5 min each, 150 mls total). After the final PBS wash, the hairsamples containing bound phage peptides were frozen at −20° C. Hairbinding peptides identified according to this example containing theconsensus sequence LEST are LESTPKMK (SEQ ID NO. 6) and LESTPKM (SEQ IDNO. 7)

Example 3 Selection of Phage-Peptides that Bind to Hair or Skin UsingPCR for Identification of High Affinity Phage-Peptide Clones

The skin swatches and hair samples were frozen at −20° C. until PCR. Inone example, PCR was performed directly on the hair and skin samplesusing the following conditions in 0.5 ml PCR tubes with the followingreagents:

50 μl reaction mix (HotStart)

2 μl CB05 primer (25 μM)

2 μl CB12 primer (25 μM)

46 μl sterile dH₂O

5 μl of BSA at 10 mg/ml and 50 μl of mineral oil were added. PCRamplification was performed post initiation at 95° C. for 15 min, using30 cycles of denaturation at 94° C. for 30 sec, annealing at 58° C. for30 sec and synthesis at 72° C. for 60 sec. Extension was preformed at72° C. for 10 min. Primers were obtained from Operon Technologies, Inc.(Alameda, Calif.). The sequences of the primers were:

SEQ ID NO. 1 CB05 CGTAGTGGCATTACGTATTTTACCCGTTTAATGG (5′ - 3′) SEQ IDNO. 2 CB12 CGAGAGGGTTGATATAAGTATAGCCCGGAATAGG (5′ - 3′)

Additionally 1 μl of the different PCR products was subjected to anotherround of PCR using the same program but the following ingredients wereadded:

50 μl reaction mix (HotStart)

1 μl CM13 01 primer (50 μM)

1 μl CM13 02 primer (50 μM)

47 μl sterile dH₂O

50 μl of BSA at 10 mg/ml and μl of mineral oil were added. Primers wereobtained from Operon Technologies, Inc. (Alameda, Calif.). The sequencesfor the primers were:

SEQ ID NO. 3 CM13 01 CCTCGAAAGCAAGCTGATAAACCG (5′-3′) SEQ ID NO. 4 CM1302 CATTCCACAGACAACCCTCATAG (5′-3′).

The PCR products were visualized on a 2% E-gel along with PCR productsfrom dilutions of the various initial phage peptide libraries (positivecontrol) and molecular weight markers, run under 65V for 40 min. 4 μl ofthe PCR products were subject to TOPO cloning and transformationaccording to standard protocol but all incubations were done for 30minutes. The individual clones were submitted to PCR (12.5 μl MasterMix, 0.1 μl each of CM13 01 and CM13 02 primers, 12.3 μl sterile waterper clone) using the same program as described above. Sequencing using 1μl of PCR product and 11 μl of g96 primer was completed at Sequetech(Mountain View, Calif.); Biotech Core, Inc (Mountain View, Calif.) orinternally using an ABI Applied Biosystem 373XL.

Example 4 Cloning of PCR Products

PCR products from the first round of selection were cloned as follows:

Vector Preparation:

10 μg of M13KE vector (New England Biolabs (NEB), Beverly, Mass.) wasdigested overnight (16 h) at 37° C. and according to NEB recommendedconditions, digestion was performed in 400 μl total volume as follows:M13KE, 10 μl; Eag 1, 10 μl; Acc65 I, 10 μl; 10×NEB buffer 3, 40 μl;100×BSA 4 μl; and dH₂O, 326 μl. The digested vector was purified usingQiagen PCR Purification Kit (Qiagen) using 30 μl of elution buffer (EB).The purified digest was stored at −20° C.

Insert Preparation:

PCR product from the first round of selection was purified using theQiagen Purification Kit and eluted in 30 μl of EB buffer. 15 μl of thepurified material was digested overnight in 100 μl total volume asfollows: PCR product, 15 μl; Eag1, 1 μl; Acc65 l, 1 μl; 10×NEB buffer 3,10 μl; 100×BSA, 1 μl; and dH₂O, 64 μl. The digestion was followed by aheat shock treatment at 60° C. for 20 min and the product was stored at−20° C. until further use.

The ligation was performed as described below using the Takara kit at16° C. for 30 min, then placed on ice. Vector preparation, 1 μl; Insertpreparation, 1 μl; EB buffer, 3 μl; and Solution 1, 5 μl from TakaraBioInc., (Shiga, Japan).

Transformation:

5 μl of ligation mixture was used to transform 50 μl of TOP10F′chemically competent cells (Invitrogen) according to the commercialprotocol. The cells were grown on LB plates overnight at 37° C.

The phage peptide libraries were amplified and titered according tostandard techniques. Subsequent rounds of deselection and selection mayalso be performed according to the methods described above.

Example 5 Enzyme-linked Assay for Selective Binding of Peptides thatTarget Human Hair and Not Skin or Target Skin and Not Hair

Peptide sequences identified in Examples 1 and 2 along with a randomcontrol peptide were C-terminally labeled with a linker molecule(biotinylated lysine) at the C-terminus (sequence GGGK (biotin)). In onespecific example, the following sequences were used: the sequenceLESTPKMK (SEQ ID NO. 6), which was screened on hair; the sequenceFTQSLPR (SEQ ID NO. 5) which was screened on skin; and an activitycontrol β-endorphin peptide YGGFMTSE (SEQ ID NO. 8).

Dark brown hair (3″ long, 4 each), moistened with 2% Neutrogena® BathGel and pre-equilibrated human skin tissues obtained from MatTek, wereplaced in the wells of a 24 well plate. 1 ml of a 200 μM solution of thebiotinylated peptide in 2% Neutrogena® Bath Gel was added to the hairand skin samples and incubated 30 min at room temperature with gentleagitation. The solution was then pipetted off and the hair and skinsamples transferred with clean tweezers to a 50 ml conical tube, washedonce with 50 ml of 2% bath gel, twice with 50 ml of water, and once with50 ml of PBS. Each wash step took 5 min and was performed on a rotaryshaker at 20 rpm. The hair and skin samples were transferred with cleantweezers to a fresh 24 well plate where 1 ml of streptavidin conjugatedhorseradish peroxidase (diluted 1/1000 in PBS) was added for 1 hr atroom temperature under gentle rocking. Excess streptavidin HRP wasremoved by washing twice with 50 ml of PBS (5 min, 20 rpm each) in a 50mL conical tube. The hair and skin samples were transferred to freshwells and 1 ml of H₂O₂/OPD (45 ml of citrate buffer at pH 4.0, onetablet of 10 mg OPD (o-phenylene diamine, Sigma Cat. # P8287) then 76 μlof H₂O₂ at 30%) solution was added. The color was left to develop atroom temperature. At various time intervals, 200 μl of solution fromeach tube was removed, placed into a 96 well MTP and absorbance was readat 430 nm. FIG. 2 shows that peptide binding is selective for therespective targets, relative to the control peptide.

What is claimed:
 1. A method for screening a peptide library comprisingthe steps of, (a) contacting said peptide library with an anti-targetunder conditions that allow binding between the peptides of said libraryand said anti-target; (b) separating anti-target unbound peptides fromanti-target bound peptides; (c) contacting said anti-target unboundpeptides with a target under conditions favorable for binding betweensaid anti-target unbound peptides and said target to form a target-boundpeptide complex; (d) separating said target-bound peptide complex fromanti-target unbound peptides which do not bind to said target; and (e)identifying the target-bound peptides on the target-bound peptidecomplex, wherein said anti-target is selected from the group consistingof skin tissue and hair, wherein when and said target is skin tissuesaid anti-target is hair and when said target is hair said anti-targetis skin tissue.
 2. The method according to claim 1, wherein steps (a),(b), (c) and (d) are repeated between 2 to 10 times using saidtarget-bound peptides.
 3. The method according to claim 1, wherein thetarget-bound peptide is not released from the target during theidentifying step.
 4. The method according to claim 1, wherein saidpeptide library is a phage display library.
 5. The method according toclaim 1, wherein separating said target-bound peptide further includesan acid elution step.
 6. The method according to claim 1, wherein step(a) and (b) are repeated between 2 to 10 times prior to said contactingstep (c).
 7. The method according to claim 1, wherein said identifiedtarget-bound peptides are less than 25 amino acids in length.
 8. Themethod according to claim 1, wherein the identified target-boundpeptides have a selectivity of binding to the target compared to bindingto the anti-target of at least 20:1.
 9. The method according to claim 4,wherein said identifying step comprises amplifying a nucleic acid codingfor said target-bound peptide in a polymerase chain reaction.